Beiging of perivascular adipose tissue regulates its inflammation and vascular remodeling

Although inflammation plays critical roles in the development of atherosclerosis, its regulatory mechanisms remain incompletely understood. Perivascular adipose tissue (PVAT) has been reported to undergo inflammatory changes in response to vascular injury. Here, we show that vascular injury induces the beiging (brown adipose tissue-like phenotype change) of PVAT, which fine-tunes inflammatory response and thus vascular remodeling as a protective mechanism. In a mouse model of endovascular injury, macrophages accumulate in PVAT, causing beiging phenotype change. Inhibition of PVAT beiging by genetically silencing PRDM16, a key regulator to beiging, exacerbates inflammation and vascular remodeling following injury. Conversely, activation of PVAT beiging attenuates inflammation and pathological vascular remodeling. Single-cell RNA sequencing reveals that beige adipocytes abundantly express neuregulin 4 (Nrg4) which critically regulate alternative macrophage activation. Importantly, significant beiging is observed in the diseased aortic PVAT in patients with acute aortic dissection. Taken together, vascular injury induces the beiging of adjacent PVAT with macrophage accumulation, where NRG4 secreted from the beige PVAT facilitates alternative activation of macrophages, leading to the resolution of vascular inflammation. Our study demonstrates the pivotal roles of PVAT in vascular inflammation and remodeling and will open a new avenue for treating atherosclerosis.

pathogenesis of vascular remodeling starts from the impaired endothelial cell function, leading to recruitment of circulating inflammatory cells to the intima-medial layer 3,6,7 . The effects of potent vascular protective agents such as estrogen, statins and microRNAs have been mostly attributed to their beneficial functions on endothelial cells [8][9][10][11][12] . There is also growing evidence suggesting that perivascular adipose tissue (PVAT), which accounts for the majority of the outer matrix surrounding systemic blood vessels, contributes to the pathophysiological vascular response in an 'outside-in' manner 13 . Adipose tissues are classified into two distinct phenotypes; white adipose tissue (WAT), which stores energy as triglycerides, and brown adipose tissue (BAT), which dissipates energy as heat 14 . A brown adipocyte-like phenotype has been reported to emerge within WAT in response to various stimuli such as cold exposure and β3-adrenergic receptor (β3AR) agonist 14,15 . This unique phenomenon, called 'browning' or 'beiging', is controlled by transcriptional regulators, such as PRDM16, NFIA and EBF2 [16][17][18][19][20][21][22] , and observed in humans as well as in rodents, contributing to energy homeostasis 14,15 . Recent basic and clinical studies reported that human PVAT surrounding coronary arteries show phenotypic changes in response to vascular inflammation, which can be detected by newly innovated computerized tomography-based methods 23,24 . Although these findings suggest an association between PVAT phenotypic changes and vascular diseases, the causality and mechanisms involved remain unclear.
In this work, we examined the roles of PVAT in vascular inflammation and subsequent pathological remodeling. We show that vascular injury induces the beiging of PVAT, which fine-tunes inflammatory response and thus vascular remodeling as a protective mechanism.

Endovascular injury induces PVAT beiging
We generated endovascular injury by inserting a wire into the femoral arteries (FAs) and examined inflammatory response in PVAT. Given the considerable effects of estrogen on the vasculature 25,26 , male or ovariectomized female mice were used in the study. Genes of various types of immune cells were upregulated in PVAT at 24 h after vascular injury, including the monocyte-macrophage marker genes, Cd11b, Cd11c, Mrc1, and Adgre1 (F4/80) (Fig. 1a). Immunohistochemical staining revealed that F4/80 + macrophages accumulated predominantly in the outer tissues surrounding the vasculature, which were mainly comprised of PVAT, rather than in the vascular tissues, at the early phase (3 days) after injury (Fig. 1b, c). These F4/80 + macrophages then spread into the vascular tissues at the late phase (14 days) after injury (Fig. 1b,  c). Concurrently, the expressions of inflammatory cytokine genes such as Tnf (TNF-α), Serpine1 (PAI-1), Il1a and Il1b, were significantly upregulated in the surrounding tissues after vascular injury (Fig. 1d).
Transcriptome analysis of PVAT surrounding injured or shamoperated FAs showed that in addition to the increase in expression levels of several genes encoding immune cell markers and inflammatory cytokines, vascular injury induced a significant increase in expression levels of BAT marker genes including Ucp1, Cidea, Cox8b, Ppargc1a, Elovl3 and Dio2 in the PVAT ( Supplementary Fig. 1a), which was confirmed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis (Fig. 1e). Upregulation of UCP1 in PVAT after vascular injury was confirmed by in situ hybridization analysis (Fig. 1f) and immunohistochemical analysis (Fig. 1g, h). Morphologically, adipocytes in PVAT after vascular injury possessed the beige/BAT characteristics including smaller cell size and multilocular lipid droplets compared with those after sham operation (Fig. 1g, i). Western blot analysis also showed the increase in UCP1 exclusively in the outer tissues denuded from vessels rather than the remained arteries (Fig. 1j). Then, we examined the role of infiltrated macrophages in the upregulation of UCP1. Reduction of macrophages by the administration of clodronate liposome significantly attenuated upregulation of UCP1 after vascular injury (Fig. 1k, l). The effects of ovariectomy on the expression levels of BAT markers, such as Ucp1 and Elovl3, were marginal, while the upregulation of these markers after vascular injury was more prominent in ovariectomized mice than in controls ( Supplementary Fig. 1b). A significant upregulation of UCP1 and the morphological shrinkage of adipocyte cell size in PVAT after vascular injury were observed in intact gonadal male mice (Supplementary Fig. 1c, d). Taken together, these results suggest that in male and ovariectomized female mice‚ vascular injury elicits the infiltration of macrophages in PVAT, which induces the change of PVAT into BATlike phenotypes, resembling the "beiging"-phenomenon that is observed in subcutaneous and visceral WAT following exposure to cold or β3AR agonists.

PVAT beiging alters vascular inflammatory response
We next investigated the pathophysiological roles of the PVAT beiging in the development of vascular remodeling after injury using mice with an adipocyte-specific deletion of Prdm16 gene, an essential regulator of beiging [16][17][18] , using Adipoq (Adiponectin)-Cre +/− ;Prdm16 fl°x/fl°x mice, referred to as Adipo Cre+ ;Prdm16. Prdm16 mRNA expression was >90% lower in adipose tissues such as thoracic aorta PVAT and intra-scapular BAT compared with Adipo Cre− ;Prdm16 control littermates, despite comparable expression in other tissues such as lung and intestine ( Supplementary Fig. 2a). Vascular injury induced more prominent remodeling accompanied by marked inflammation and suppressed beiging in the PVAT in both males and ovariectomized females of Adipo Cre+ ;Prdm16 mice compared with Adipo Cre− ;Prdm16 mice at 14 days after injury (Fig. 2a, b and Supplementary Fig. 2b, c). To rule out the effects of Prdm16 deletion in adipose tissues of the whole body, we locally repressed Prdm16 by applying pluronic gel containing small interfering RNA (siRNA) against Prdm16 around FAs 27,28 . A significant reduction of Prdm16 mRNA expression was confirmed in the outer tissues surrounding FA of wild type mice after siRNA treatment, whereas no difference was observed in contralateral untreated FA ( Supplementary Fig. 2d). The Prdm16 siRNA treatment abolished PVAT beiging after vascular injury ( Supplementary Fig. 2e) and exacerbated pathological intimal thickening 14 days after vascular injury (Fig. 2c).
Macrophages change their phenotypical and functional characteristics, termed macrophage polarization, in response to pro-or anti-inflammatory stimuli 29 . Although the accumulation of F4/80 + cells was comparable between locally beiging-suppressed FA and control FA (Fig. 2c), the ratio of inducible nitric oxide synthase (iNOS) + activated inflammatory macrophages to CD206 + alternatively activated antiinflammatory macrophages was significantly increased in the beigingsuppressed FA PVAT as compared with that of control FA PVAT 14 days after vascular injury (Fig. 2c). These significant differences were not observed during the early phase (3 days) after injury ( Supplementary  Fig. 3a, b), suggesting that inhibition of PVAT beiging exacerbates pathological remodeling after vascular injury by prolonged activation of pro-inflammatory macrophages.
The effects of systemic activation of WAT beiging by exposure to cold or β3AR agonists on vascular pathogenesis in vivo remain controversial and depend on the experimental models studied 30,31 . These conflicting results are considered to come from direct and/or indirect effects of systemic activation of beiging on vascular remodeling, such as brown/beige fat-mediated lipolysis, release of anti-inflammatory adipokines and glucose homeostasis [30][31][32] . Therefore, we locally activated beiging of the FA PVAT using pluronic gel containing the specific β3AR agonist, CL316243 and evaluated the specific effects of PVAT beiging on the phenotypic transition of macrophage and vascular remodeling. Local β3AR activation significantly induced PVAT beiging in the outer surrounding tissue of FA, but not in the contralateral untreated FA, visceral WAT or BAT ( Supplementary Fig. 3c), and significantly attenuated vascular intimal thickening 14 days after injury with a shift of macrophage polarization to anti-inflammatory phenotype in FA PVAT ( Fig. 2d and Supplementary Fig. 3d). These significant differences were not observed during the early phase after injury ( Supplementary Fig. 3a, b). In contrast, the beiging in PVAT elicited by vascular injury was attenuated by a β3AR antagonist (SR59230A) with pluronic gel (Supplementary Fig. 3e). These data suggest that pharmacological beiging stimulation exerts additional protection against pathological remodeling after vascular injury by suppression of prolonged activation of pro-inflammatory macrophages, and that the beiging in PVAT after the injury is mediated by the β3AR signaling.
Then, we examined the interactions between PVAT-derived beige adipocytes and macrophages. When RAW 264.7 macrophage cells or bone marrow-derived macrophages (BMDMs) were cultured in medium conditioned by beige adipocytes differentiated from the stromal  Supplementary Fig. 3f). We further examined the effects of PVAT beiging on cell growth of macrophages. Culture medium conditioned by PVAT-derived beige adipocytes significantly decreased the number of RAW 264.7 macrophages activated by interferon-gamma (IFNγ) and lipopolysaccharide (LPS), but not that of macrophages alternatively activated by interleukin (IL) 4 ( Fig. 3g and Supplementary Fig. 3g). Culture medium conditioned by PVAT-derived adipocytes with knockdown of Prdm16 did not decrease the number of classically activated macrophages induced by IFNγ and LPS (Fig. 3g). Taken together, these results suggest that factors secreted from PVATderived beige adipocytes selectively inhibit the growth of classically activated macrophages and shift macrophage phenotypes into an alternatively activated state, resulting in anti-inflammatory effects.

NRG4 as an anti-inflammatory factor
Some studies have shown the contributions of UCP1 to atherogenesis 30,[32][33][34][35] . Therefore, we first examined UCP1 as a possible regulator of vascular remodeling after injury. Unexpectedly, vascular wall thickening 14 days after injury was comparable between Ucp1 −/− and littermate wild type (Ucp1 +/+ ) mice ( Fig. 3h), suggesting that UCP1 is not involved in the beneficial effects of PVAT beiging on vascular remodeling.
To identify the key factors that control endovascular injuryinduced inflammation in PVAT, we performed transcriptional RNA profiling using single-cell RNA sequencing (scRNA-seq) and subsequent computational analysis using publicly available scRNA-seq datasets of murine inguinal WAT (iWAT) treated with CL316243 or control (GSE 133486) 36 . Cluster analysis of uniform manifold approximation and projection (UMAP) dimensionality reduction after integration with Seurat revealed that the cells of iWAT were classified into 15 clusters (C0-14) and that C7 (Seurat) mostly consisted of CL316243treated cells (Fig. 4a, b and Supplementary Fig. 4a). Beige/brown adipocyte-specific genes such as Ucp1, Cidea and Ppargc1a, were predominantly expressed in C7 (Seurat) (Fig. 4c and Supplementary Figs. 4b, 5), indicating that this cluster represents beige adipocytes induced by β3AR activation. The emergence of the beige adipocyte cluster by β3AR activation with CL316243 was also confirmed using another integration method, Harmony (C6 (Harmony) , Supplementary Figs. 6a-f and 7). In addition, the cell type deconvolution analysis of the bulk RNA-seq dataset of iWAT (GSE129083) 37 showed that the beige adipocyte population appeared in iWAT treated with CL316243 (Supplementary Table 2). Furthermore, the analysis of another dataset (GSE 133486) 36 demonstrated that β3AR activation with cold stimuli also induced the emergence of a beige adipocyte population (C9 (cold) ), Supplementary Figs. 8a-e and 9). Gene ontology analysis for the significantly regulated genes in C7 (Seurat) identified Nrg4 as a highly expressed secretory factor involved in the molecular function of receptor-binding (Fig. 4c, d). The enrichment of Nrg4 was also detected in C6 (Harmony) and C9 (cold) (Supplementary Figs. 6d, e and 8c, d).
Consistent with the findings from the comprehensive data analysis, we observed that Nrg4 was upregulated in PVAT after vascular injury (Fig. 4e) and in cultured PVAT-derived preadipocytes stimulated by beige adipocyte differentiation factors, accompanied by other beige genes in a time-dependent manner (Fig. 4f). The upregulation of Nrg4 in PVAT surrounding injured FAs was also observed 14 days after injury by in situ hybridization analysis ( Supplementary Fig. 10a), where Nrg4 was expressed in beige adipocytes of PVAT expressing Ucp1 ( Supplementary Fig. 10b). Nrg4 upregulation was significantly attenuated in Adipo Cre+ ;Prdm16 mice and PVAT-derived preadipocytes transduced with siRNA against Prdm16 or Nfia (Fig. 4g, h), whereas it occurred independently of UCP1 ( Supplementary Fig. 10c). NRG4 has recently emerged as a brown fat-enriched secreted factor that ameliorates diet-induced metabolic disorders, including insulin resistance and hepatic steatosis [38][39][40] . We thus examined whether NRG4 was involved in beige adipocytes-induced resolution of macrophage inflammation. Culture media conditioned by PVAT-derived beige adipocytes induced phenotypic changes from classical to alternative activation and downregulation of inflammatory genes in RAW 264.7 macrophages or BMDMs, but these changes were not observed by the culture media conditioned by PVAT-derived beige adipocytes with knockdown of Nrg4 by siRNA ( Supplementary Fig. 10d and Fig. 4i-l). Consistently, recombinant murine NRG4 attenuated the Cd86/Mrc1 ratio and inflammatory cytokine levels in macrophages classically activated ( Supplementary Fig. 10e, f). The knockdown of Erb-B2 receptor tyrosine kinase 4 (ErbB4), a receptor of NRG4 41 , in macrophages abolished the alternative activation and anti-inflammatory effects of culture media conditioned with PVAT-derived beige adipocytes ( Supplementary Fig. 10g-i). Furthermore, culture media conditioned by PVAT-derived beige adipocytes with Nrg4 knockdown no longer inhibited the increase in the number of RAW 264.7 cells activated by IFNγ and LPS (Fig. 4m). In mice, Nrg4 siRNA treatment with pluronic gel was found to exacerbate pathological intimal thickening 14 days after vascular injury (Fig. 4n). Taken together, these results suggest that NRG4 secreted from PVAT-derived beige adipocytes Fig. 1 | Endovascular injury-induced macrophage accumulation and inflammation in PVAT followed by upregulation of BAT markers. a Gene expressions of immune cell markers in PVAT 24 h after vascular injury (sham, n = 4 for Cd8, 6 for others; injury, n = 9, two-tailed t tests with Holm-Sidak's correction for multiple comparisons). b Haematoxylin and eosin (H&E), Elastica van Gieson (EVG) and immunohistochemical staining for F4/80 in the early (day 3) and late (day 14) phases after injury. Blue dashed lines indicate the external elastic lamina. Scale bars represent 100 μm (thick bars) and 50 μm (thin bars). Images are representative of three independent experiments. c Time course of F4/80 + cell accumulation in arteries (vessel) and outer tissues mainly composed of PVAT (n = 3, 4, 7, 7, 6 (from left to right) at each group, respectively, one-way analysis of variance (ANOVA) and Tukey-Kramer post-hoc test). and immunohistochemical staining for UCP1 in injured (3 and 14 days after injury) or sham-operated FAs and outer tissue. Scale bars represent 100 μm (thick bars) and 50 μm (thin bars). Images are representative of three independent experiments. h Time course of UCP1 + cells accumulation in PVAT (n = 3, 4, 7, 7, 6 at each group, respectively, one-way ANOVA followed by Tukey-Kramer post-hoc test). i Histograms of adipocyte area of sham-operated or injured PVAT (14 days after injury). Three images of each biological replicate were analyzed and combined to create the histogram. The size distribution between each group was compared using Kolmogorov-Smirnov test. Each bin was normalized to a percent of the total count for that individual tissue. Adipocytes of the bin size in the range of 20-500 μm 2 were included for the analysis. j Representative images of western blots for UCP1 in arteries (vessel) and outer tissues mainly composed of PVAT harvested 48 h after injury. Intra-scapular BAT was used as a positive control. GAPDH was used for internal control (n = 4 for each group, representative images are shown). k Immunohistochemical staining for F4/80 and UCP1 in outer tissue surrounding FAs 14 days after injury was performed in mice treated with either clodronate or vehicle. Scale bars represent 50 μm. Images are representative of three independent experiments. l F4/80 + and UCP1 + area in PVAT (vehicle, n = 9; clodronate, n = 4, unpaired two-tailed Student's t test). Data represent mean ± SEM. Source data are provided as a Source Data file.
inhibits the growth of classically activated macrophages and induces phenotypic shift to an alternatively activated state, leading to accelerated resolution of macrophage inflammation, resulting in attenuation of pathological vascular remodeling after injury.

PVAT beiging is detectable in human AAD
Acute aortic dissection (AAD) is the acute destruction of aortic wall that accompanies severe local inflammation 42 . We examined whether beiging was recognized in PVAT of human aorta with AAD. In addition to the accumulation of macrophages, protein expression levels of markers for beige cells, such as UCP1 and CIDEA, and NRG4 were significantly increased likely as a compensatory response in the PVAT of AAD lesions compared with the PVAT without AAD (Fig. 5a, b and Supplementary Fig. 11a, b). To elucidate the role of PVAT beiging in AAD, we used an AAD murine model with the systemic administration of angiotensin II, β-aminopropionitrile, and N-nitro-L-arginine methyl ester [43][44][45] . CL316243 administration significantly reduced death due to dissection or rupture of the aorta (Fig. 5c), concomitant with the shift  Supplementary Fig. 12). These findings suggest that beiging occurs in the PVAT of the human aorta during acute dissection and may regulate the inflammatory response during the development of AAD.

Discussion
Vascular damage provokes regional inflammation and prolonged inflammation leads to pathological vascular remodeling 4,5 . The balance of pro-and anti-inflammation is critical for successful vascular wound healing and clinical outcomes 46,47 . The primary process of inflammation resolution is accomplished by the phenotypic conversion of proinflammatory macrophages into anti-inflammatory macrophages 47 . However, the mechanisms involved in this conversion have not yet been fully elucidated. This study revealed that endovascular injury induced 'beiging' of regional PVAT, and that inhibition of PVAT beiging exacerbated thickening of the intima after injury accompanied by an increased accumulation of pro-inflammatory macrophages, while activation of beiging conversely attenuated pathological vascular remodeling and accumulation of pro-inflammatory macrophages. These findings suggest that beiging of PVAT plays a pivotal role in changing the initial inflammation phase to its resolution after vascular injury. Various studies have suggested that endothelial dysfunction leads to the recruitment of circulating monocytes to the intima-medial layer 3,6 . In our study, the marked accumulation of monocytes was also identified in the outer tissues surrounding the vasculature from the early phase after vascular injury. The infiltrated monocyte-macrophages elicited PVAT beiging, resulting in the suppression of the excessive vascular inflammatory response to the injury. These data indicate a pivotal role of the "outside-in" manner in wound healing after vascular injury. Local activation of PVAT beiging attenuated pathological vascular remodeling after injury, suggesting that PVAT acts directly on nearby arteries and mediates the pathophysiological response to vascular damage in a paracrine manner. We identified NRG4 as an anti-vascular remodeling factor derived from beige PVAT. NRG4 is a member of the NRG protein family, which acts via ErbB receptor tyrosine kinases. This molecule is highly expressed in the pancreas, skeletal muscles and BAT 48 and protects against diet-induced insulin resistance and hepatic steatosis through regulating hepatic lipogenic and cytoprotective signaling 38,40 . Recent studies have shown the expression of functional ErbB receptors on innate immune cells such as macrophages, dendritic cells and neutrophils 49 , and the NRG4-ErbB4 axis exerts anti-inflammatory effects in macrophages by promoting apoptosis of classically activated macrophages but not alternatively activated macrophages 49,50 . Consistent with these findings, medium conditioned by PVAT-derived beige adipocytes significantly inhibited the number of classically activated macrophages, which was abolished by Nrg4 knockdown. Reduced levels of NRG4 have been reported to be associated with increased carotid intimal thickness, increased angiographic severity of coronary artery disease and acute coronary syndrome [51][52][53] . In this study, PVAT beiging was detected in human aorta with acute dissection, suggesting that NRG4 secreted from beige PVAT might protect human aorta against AAD. The lineage of the beige cells that emerged in the PVAT was not examined in this study. Reportedly, smooth muscle cell has been considered a possible lineage of origin for PVAT 54 , and Angueira et al. have shown an increase in Myh11 + smooth muscle cell-derived adipocytes in thoracic PVAT after treatment with rosiglitazone 55 . Since rosiglitazone reportedly recruits beige cells in fat tissues 56 , the beige cells in PVAT may be originated from smooth muscle cells. Lineage tracing studies using inducible genetic techniques may provide further biological and functional insights into the beiging phenomenon in the PVAT. Our data showed that the beiging after injury was suppressed by the macrophage depletion or the local β3AR inhibition, implying that the PVAT beiging was elicited via β3AR signaling mediated by infiltrated macrophages. Meanwhile, the involvement of other signaling pathways of beiging such as succinate metabolisms and BMP4 in the beiging process in PVAT after injury remains to be elucidated 57,58 . In conclusion, the present study revealed that PVAT beiging plays a critical role in the vascular inflammatory response to injury by controlling macrophage inflammation and its resolution and suggests that PVAT beiging and NRG4 may be novel targets of vascular injury including AAD.

Animal studies
All animal procedures were approved by the University of Tokyo Ethics Committee for Animal Experiments and strictly adhered to the guidelines for animal experiments at the University of Tokyo. Male or, unless otherwise specified, ovariectomized female mice were used for the study. All wild type C57BL/6J mice were purchased from CLEA Japan. Adipo Cre+ mice (B6;FVB-Tg(Adipoq-Cre)1Evdr/J, #10803), Prdm16 flox/flox mice (B6.129-Prdm16 tm1.1Brsp /J, #024992) and Ucp1 −/− mice (B6.129-Ucp1 tm1Kz /J, #003124) were purchased from Jackson Laboratory. Conditional deletion of Prdm16 in adipocytes was achieved by crossing Prdm16 flox/flox homozygous mice with Adipo Cre+ hemizygous mice. Genotyping PCR was performed according to the protocol from Jackson Laboratory. All mice were maintained in specific pathogen-free conditions in the animal facilities of the University of Tokyo. They were housed in a controlled environment with a 12 h light/12 h dark cycle at a maintained temperature and kept with free access to food and water throughout the whole experiment period. Ambient room temperature was regulated at 73 ± 5°F and humidity was controlled at 50 ± 10%. Endovascular injury of femoral arteries (FA) with wire insertion was performed at 10-12 weeks of age using a straight spring wire (0.38 mm in diameter, COOK) as previously described 59 . Arteries and outer tissues surrounding the vasculature were collected. Ovariectomies were performed in 8-10-week-old female mice 2 weeks before the endovascular injury, as previously described 60 . To selectively remove the macrophages, mice were given intraperitoneally clodronate liposomes Fig. 2 | Modification of PVAT beiging alters vascular inflammatory response and remodeling after injury. a EVG staining in FAs 14 days after injury of Adipo Cre+ ;Prdm16 mice and Adipo Cre− ;Prdm16 (control) mice. The ratio of intima to media area (intima/media) and the % intima-medial area in the area surrounded by the external elastic lamina (area of intima-media) at 14 days after vascular injury were analyzed (Adipo Cre− ;Prdm16, n = 10; Adipo Cre+ ;Prdm16, n = 13, unpaired two-tailed Student's t test). Scale bars represent 100 μm. b Gene expression of inflammatory cytokine markers in PVAT 14 days after vascular injury in Adipo Cre+ ;Prdm16 mice and Adipo Cre− ;Prdm16 (control) mice (Adipo Cre-;Prdm16sham, n = 6; Adipo Cre+ ;Prdm16sham, n = 7; Adipo Cre-;Prdm16injury, n = 6; Adipo Cre+ ;Prdm16injury, n = 6, one-way ANOVA followed by Tukey-Kramer post-hoc test). c, d EVG and immunohistochemical staining for F4/80, iNOS (classically activated) and CD206 (alternatively activated) in FAs 14 days after injury in wild type mice treated with pluronic gel containing siRNA [Prdm16 or scrambled, (c)] or β3AR agonist [CL316243 or vehicle, (d)] applied to the PVAT surrounding the FA. The ratio of intima to media area (intima/media) and the % intima-medial area in the area surrounded by the external elastic lamina (area of intima-media) at 14 days after vascular injury were analyzed ((c) scrambled siRNA, n = 12; Prdm16 siRNA, n = 11, unpaired two-tailed Student's t test, (d) vehicle, n = 11; CL316243, n = 9, unpaired two-tailed Student's t test). The positive area of immunostaining and ratio of iNOS to CD206-positive area in arteries (vessel) or surrounding tissues (PVAT) were analyzed (c scrambled siRNA, n = 16; Prdm16 siRNA, n = 15, unpaired two-tailed Student's t test, d vehicle, n = 11; CL316243, n = 9, unpaired two-tailed Student's t test). Scale bars represent 100 μm. Data represent mean ± SEM. Source data are provided as a Source Data file.

Local administration of reagents
Pluronic F-127 gel (Sigma-Aldrich) is a thermoreversible gel 63 , which is liquid at refrigerated temperatures (4-5°C), but gel upon warming to room temperature 64 . The gelation is reversible upon cooling. For local gene knockdown studies, 0.2 nmol/mouse Ambion in vivo predesigned siRNA against Prdm16 (s89042), Nrg4 (s206928), or  negative control mixed with Invivofectamine 3.0 reagent (Thermo Fisher Scientific) was dissolved in pluronic gel on ice and administered locally in PVAT of the injured artery. For β3AR signal activation or inhibition studies, β3AR agonist (CL316243, 0.01 mg/kg; Sigma-Aldrich), β3AR antagonist (SR59230A, 0.02 mg/kg; Sigma-Aldrich), vehicle PBS, or vehicle dimethyl sulfoxide was dissolved in pluronic gel and administered in the PVAT.

Single-molecule fluorescence in situ hybridization
In situ hybridization was performed using the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics) according to manufacturers' instructions. The probes targeting mm-Ucp1 (#455411) or mm-Nrg4 (#493731, Advanced Cell Diagnostics) were hybridized, followed by RNAscope amplification and co-staining with fluoresceinconjugated WGA to detect cell borders. Slides were mounted with ProLong Diamond Antifade Mountant with DAPI (Life Technologies). Fluorescent signals were captured with the ×40 objective lens on a laser scanning confocal microscope (LSM880, Zeiss). The number of positive dots was calculated using NIH ImageJ.

PVAT-derived preadipocyte isolation
Eleven-week-old male wild type mice (for fluorescence-activated cell sorting analysis) or twelve-week-old female wild type mice (for cell culture) were sacrificed, and the PVAT from the thoracic aorta were extracted. PVAT was minced into small pieces using sharp round scissors and digested using gentleMACS (Miltenyi Biotec) and Adipose Tissue Dissociation Kit (Miltenyi Biotec), as previously described 68 .
Digestion was performed in a MACSmix Tube Rotator (Miltenyi Biotec) at 37°C for 40 min.

Immortalization of PVAT-derived preadipocyte
A stromal vascular fraction of PVAT from 12-week-old female wild type mice was isolated and cultured in collagen-coated dishes, as previously described 68 . Preadipocytes isolated from the PVAT stromal vascular fraction were immortalized using the SV40 antigen as described previously 19 .

BMDM isolation
Bone marrow cells were flushed from the femurs and tibias of 10-weekold male wild type mice, and cultured at 37°C in DMEM/F12 containing 1% streptomycin, 1% penicillin, and 10% fetal bovine serum (FBS) (Cosmo Bio) and in 40 ng/mL of recombinant mouse M-CSF (576406; BioLegend) for 7 days, as previously described 69 . The differentiated BMDMs were detached from plates using TrypLE (Thermo Fisher Scientific) and replated into 12-well tissue culture dishes at a density of 5 × 10 5 cells per well prior to cell stimulation.  followed by Tukey-Kramer post-hoc test). i-l PVAT-derived preadipocytes were introduced by Nrg4 or scrambled siRNA and stimulated with beige differentiation factors. Culture media conditioned by these cells were then added to RAW 264.7 cells (i, j) or BMDMs (k, l) and mRNA was extracted 48 h after treatment. The results of qRT-PCR for the gene expression levels of macrophage phenotype markers (i, k) and inflammatory cytokines (j, l) are shown ((i, j) n = 4 biological replicates, representative data of three different culture lines are shown, two-tailed t tests with Holm-Sidak's correction for multiple comparisons, (k, l) n = 3 biological replicates, representative data of three different culture lines are shown, two-tailed t tests with Holm-Sidak's correction for multiple comparisons). m Cell growth analysis of RAW 264.7 cells 72 h after treatment with conditioned media from PVAT-derived beige adipocytes. RAW 264.7 cells were pre-treated with IFNγ and LPS or IL4 for 24 h and treated with media cultured in the absence of adipocytes (gray dots), media conditioned by adipocytes (red dots) and media conditioned by adipocytes introduced by Nrg4 siRNA (blue dots) (n = 4 biological replicates, representative data of three different culture lines are shown, one-way ANOVA followed by Tukey-Kramer posthoc test). n EVG staining in FAs 14 days after injury in wild type mice treated with pluronic gel, containing siRNA against Nrg4 or scrambled, that was applied to the PVAT surrounding the FA. The ratio of intima to media area (intima/media) and the % intima-medial area in the region surrounded by the external elastic lamina (area of intima-media) at 14 days after vascular injury were analyzed (scrambled siRNA, n = 10; Nrg4 siRNA, n = 11, unpaired two-tailed Student's t test). Scale bars represent 100 μm. Data represent mean ± SEM. Source data are provided as a Source Data file.

Differentiation of PVAT stromal vascular fraction to beige cells
PVAT-derived preadipocytes were differentiated to beige cells according to the previous reports 68 36 , the data from Adipo Cre-;Il10ra flox/flox mice were used for the present analysis. The standard Seurat (V.4.0.1) for R (V.4.0.2) protocol ("FindIntegrationAnchors" and "IntegrateData") was used for the integration 73 . To observe the effects of CL316243 treatment, an alternative integration step was performed with Harmony (V.0.1) 74 . All sequenced cells were projected onto two dimensions using UMAP on R v.4.0.2. The optimal number of principal components used for UMAP dimensionality reduction was determined using the Jackstraw permutation approach and a grid-search of the parameters. Cells were divided into several clusters and color-coded according to cell types. Cells were also color-coded for the treatment group (CL316243 or cold stimulation) (pink) or control (turquoise). Marker genes were searched using the FindMarkers function in Seurat. Gene Ontology analysis was performed using Database for Annotation, Visualization, and Integrated Discovery ver. 6.8 (DAVID 6.8) 75 . Furthermore, the gene signature from the scRNA-seq data was used to deconvolute public bulk RNA-seq data to confirm the validity of the cell type clustering performed in this scRNA-seq re-analysis. We confirmed that the beige adipocytes also exist in iWAT treated with CL316243 in another public bulk RNA-seq study (GSE129083) 37 using MuSiC, a method for predicting cell abundance based on single-cell data 76 .

Statistics and reproducibility
Data were presented as means ± standard error of mean (SEM). All statistics are described in figure legends. In general, comparisons between two groups were performed using the two-tailed Student's t test or Mann-Whitney U-test, and multiple group comparisons were performed by one-way ANOVA followed by Tukey-Kramer post-hoc test. Survival curves were created using the Kaplan-Meier method and compared by a log-rank test. All statistical analyses were performed using the Prism software (GraphPad). P-values of <0.05 were considered statistically significant.

Human studies
Pathological records were reviewed, and formalin-fixed paraffinembedded descending aorta tissues were obtained from the archives of the Department of Pathology, The University of Tokyo Hospital. The aortic specimens of AAD were obtained by the autopsy of six patients who died of AAD, five males and one female, aged between 45 and 84 years; four of these patients had hypertension, one patient had suffered from a previous stroke, and three had a smoking history. The aortic specimens of controls were autopsy specimens from five patients who died of non-aortic causes, four males and one female, aged between 44 and 78 years; one patient had hypertension, one patient suffered from dyslipidemia, one with chronic kidney disease, one suffered from a previous stroke, and one had a smoking history. Patients with an apparent history of inherited aortic diseases such as Marfan syndrome were not included in the AAD cases and controls. The inclusion and exclusion criteria for patients with AAD were as follows: AAD cases were selected from consecutive pathological autopsies from 2006 to 2017 with a description of aortic dissection as a finding in autopsy report. Controls were selected from consecutive pathological autopsies from 2014 to 2017 with no significant changes in the aortic wall based on the autopsy. The immunostaining using primary antibodies against CD204 (Transgenic, KT022, 1:500) was performed using a Ventana Benchmark automated stainer (Ventana Medical Systems). The study followed the principles outlined in the Declaration of Helsinki. This study was approved by the ethics committee of the University of Tokyo (approval ID-2020019NI), and written informed consent was waived because this is a retrospective study using existing tissue blocks. Instead, we used an opt out approach to provide participants with an informed choice about participation, although no patient in the cohort for screening used the opt out option.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Source data are provided with this paper. The RNA-seq data have been deposited in GSE 206399, GSE133486, and GSE129083. The information of mouse genome (mm9) is available on UCSC Genome Browser (http://genome.ucsc.edu). The authors declare that all data are available in the article file and Supplementary information files, or available from the authors upon reasonable request. Source data are provided with this paper.